Inherited retinal degenerations (IRDs), with an overall global prevalence of 1/2,000 (1), are a major cause of blindness worldwide. Among the most frequent and severe IRDs are retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), and Stargardt disease (STGD), which are most often inherited as monogenic conditions. The majority of mutations causing IRDs occur in genes expressed in neuronal photoreceptors (PR), rods and/or cones in the retina (2). No therapy is currently available for these blinding diseases.
Gene therapy holds great promise for the treatment of IRDs. Among the available gene transfer vectors, those based on the small adeno-associated virus (AAV) are most efficient at targeting both PR and retinal pigment epithelium (RPE) (3-4) for long-term treatment upon a single subretinal administration (3-4). Recently the inventors and others, have demonstrated that subretinal administration of AAV is well-tolerated and effective for improving vision in patients affected with type 2 LCA, which is caused by mutations in RPE65, a gene expressed in the RPE (5-9). These results bode well for the treatment of other forms of LCA and IRDs in general. The availability of AAV vector serotypes such as AAV2/8, which efficiently targets PR (10-14) and RPE, further supports this approach. However, a major limitation of AAV is its cargo capacity, which is thought to be limited to around 5 kb, the size of the parental viral genome (15-19). This limits the application of AAV gene therapy approaches for common IRDs that are caused by mutations in genes whose coding sequence (CDS) is larger than 5 kb (herein referred to as large genes). These include:
DISEASEGENECDSEXPRESSIONStargardt DiseaseABCA4 6.8 Kbrod&cone PRsUsher 1BMYO7A 6.7 KbRPE and PRsLeber CongenitalCEP290 7.5 Kbmainly PRs (pan retinal)Amaurosis10Usher1D, NonsyndromicCDH2310.1 KbPRsdeafness, autosomalrecessive (DFNB12)Retinitis PigmentosaEYS 9.4 KbPR ECMUsher 2AUSH2a15.6 Kbrod&cone PRsUsher 2CGPR9818.0 Kbmainly PRsAlstrom SyndromeALMS112.5 Kbrod&cone PRs
Stargardt disease (STGD; MIM#248200) is the most common form of inherited macular degeneration caused by mutations in the ABCA4 gene (CDS: 6822 bp), which encodes the all-trans retinal transporter located in the PR outer segment (20); Usher syndrome type IB (USH1B; MIM#276900) is the most severe form of RP and deafness caused by mutations in the MYO7A gene (CDS: 6648 bp) (21) encoding the unconventional MYO7A, an actin-based motor expressed in both PR and RPE within the retina (22-24).
Cone-rod dystrophy type 3, fundus flavimaculatus, age-related macular degeneration type 2, Early-onset severe retinal dystrophy, and Retinitis pigmentosa type 19 are also associated with ABCA4 mutations (ABCA4-associated diseases).
Various strategies have been investigated to overcome the limitation of AAV cargo capacity. Several groups, including the inventors' own, have attempted to “force” large genes into one of the many AAV caspids available by developing the so-called oversize vectors (25-27). Although administration of oversize AAV vectors achieves therapeutically-relevant levels of transgene expression in rodent and canine models of human inherited diseases (27-30), including the retina of the Abca4−/− and shaker 1 (sh1) mouse models of STGD and USH1B (27, 30), the mechanism underlying oversize AAV-mediated transduction remains elusive. In contrast to what the inventors and others originally proposed (25-27), oversize AAV vectors do not contain a pure population of intact large size genomes but rather a heterogeneous mixture of mostly truncated genomes≤5 kb in length (15-18). Following infection, reassembly of these truncated genomes in the target cell nucleus has been proposed as a mechanism for oversize AAV vector transduction (15-17, 31). Independent of transduction mechanism and in vivo efficacy, the heterogeneity in oversize AAV genome sizes is a major limitation for their application in human gene therapy.
Alternatively, the inherent ability of AAV genomes to undergo intermolecular concatemerization (32) is exploited to transfer large genes in vivo by splitting a large gene expression cassette into halves (<5 kb in size), each contained in one of two separate (dual) AAV vectors (33-35). In the dual AAV trans-splicing strategy, a splice donor (SD) signal is placed at the 3′ end of the 5′-half vector and a splice acceptor (SA) signal is placed at the 5′ end of the 3′-half vector. Upon co-infection of the same cell by the dual AAV vectors and inverted terminal repeat (ITR)-mediated head-to-tail concatemerization of the two halves, trans-splicing results in the production of a mature mRNA and full-size protein (33). Trans-splicing has been successfully used to express large genes in muscle and retina (36-37).
In particular, Reich et al. (37) used the trans-splicing strategy with AAV2 and AAV5 capsids and show that both vectors transduce both retinal pigment epithelium and photoreceptors using LacZ gene as a reporter gene. This strategy was not employed using a therapeutic and/or large gene.
Alternatively, the two halves of a large transgene expression cassette contained in dual AAV vectors may contain homologous overlapping sequences (at the 3′ end of the 5′-half vector and at the 5′ end of the 3′-half vector, dual AAV overlapping), which will mediate reconstitution of a single large genome by homologous recombination (34). This strategy depends on the recombinogenic properties of the transgene overlapping sequences (38). A third dual AAV strategy (hybrid) is based on adding a highly recombinogenic region from an exogenous gene [i.e. alkaline phosphatase, AP (35, 39)] to the trans-splicing vector. The added region is placed downstream of the SD signal in the 5′-half vector and upstream of the SA signal in the 3′-half vector in order to increase recombination between the dual AAVs. The document US2010/003218 is directed to an AP-based hybrid dual vector system. The document shows the transduction efficiency of the AP-based hybrid dual vector expressing mini-dystrophin but no data concerning efficacy.
Lopes et al. (30) studied retinal gene therapy with a large MYO7A cDNA using adeno-associated virus and found that MYO7A therapy with AAV2 or AAV5 single vectors is efficacious to some extent, while the dual AAV2 approach proved to be less effective.
Therefore there is still the need for constructs and vectors that can be exploited to reconstitute large gene expression for an effective gene therapy.